Natural‐abundance isotope ratio mass spectrometry as a means of evaluating carbon redistribution during glucose–citrate cofermentation by Lactococcus lactis

Abstract

The cometabolism of citrate and glucose by growing Lactococcus lactis ssp. lactis bv. diacetylactis was studied using a natural-abundance stable isotope technique. By a judicious choice of substrates differing slightly in their 13 C/ 12 C ratios, the simultaneous metabolism of citrate and glucose to a range of compounds was analysed. These endproducts include lactate, acetate, formate, diacetyl and acetoin. All these products have pyruvate as a common intermediate. With the objective of estimating the degree to which glucose and citrate metabolism through pyruvate may be differentially regulated, the d 13 C values of the products accumulated over a wide range of concentrations of citrate and glucose were compared. It was found that, whereas the relative accumulation of different products responds to both the substrate concentration and the ratio between the substrates, the d 13 C values of the products primarily reflect the availability of the two substrates over the entire range examined. It can be concluded that in actively growing L. lactis the maintenance of pyruvate homeostasis takes precedence over the redox status of the cells as a regulatory factor.Keywords: carbon balance; isotope ratio mass spectrometry; lactic acid bacteria; metabolic regulation; pyruvate.A range of simple sugars can be catabolized anaerobically by Lactococcus lactis and other lactic acid bacteria (LAB) in order to obtain energy for growth. Central to this metabolism is the C3 compound, pyruvate [1]. This metabolite forms the link between the essentially oxidative reactions of energy production and those required for the regeneration of reducing equivalents NAD + or NADP + (Fig. 1). However, pyruvate is relatively toxic [1,2], necessitating strict control over the level to which it accumulates. In

Liquid chromatography combined with mass spectrometry for 13C isotopic analysis in life science research

“…Nowadays, stable isotopes are used in many scientific disciplines, such as ecobiology (Moens, Bouillon, & Gallucci, 2005), geochemistry (Craig, 1953; Hayes et al, 1990), drug manufacturing (Jasper et al, 2004), forensic science (Benson et al, 2006), doping control (Ayotte et al, 2001; Aguilera, Hatton, & Catlin, 2002), authenticity assessment (Hor et al, 2001), environmental chemistry (McRae et al, 2000; Schmidt et al, 2004), and even in art (Fortunato, Ritter, & Fabian, 2005). In the domain of life sciences, the commercial availability of labeled precursors has increased the number of stable isotope applications in various fields such as, for example, medicine (Ando & Tanaka, 2005), nutrition (Dolnikowski et al, 2005), toxicology (Turner, 2006), metabolic studies (Berthold et al, 1991; Mahmoud, Gentil, & Robins, 2004), and in quantitative proteomics (Doherty et al, 2005; MacCoss & Matthews, 2005).…”

Determination of the natural abundance δ15N of nicotine and related alkaloids by gas chromatography/isotope ratio mass spectrometry

“…It can also be used to follow the metabolism of more than one pool of metabolite simultaneously. 8 An advantage of using d 15 N values rather than d 13 C values is that, in some molecular species, including nicotine 9 and 3,4-methylenedioxymethamphetamine (ecstasy), 10 the d 15 N has a wider range of values than the d 13 C, thus increasing the ease of discrimination between different sources. It is also better adapted to following the metabolism of nitrogenous molecules.…”

“…Isotope ratio mass spectrometry (IRMS) permits isotopic determination at natural abundance or at low levels of enrichment, making it an excellent choice for studies in which enrichment is difficult, expensive or prohibited. It can also be used to follow the metabolism of more than one pool of metabolite simultaneously 8. An advantage of using δ 15 N values rather than δ 13 C values is that, in some molecular species, including nicotine9 and 3,4‐methylenedioxymethamphetamine (ecstasy),10 the δ 15 N has a wider range of values than the δ 13 C, thus increasing the ease of discrimination between different sources.…”